Our research efforts this past year were directed at elucidating the mechanisms underlying the trafficking of glutamate receptors and related proteins to the postsynaptic density. Much of the research has focused on NMDA receptors and proteins which associate with them. Since the NMDA receptor performs a critical function at the synapse, it is important to understand how this receptor is delivered to the synapse and how the number and composition of receptors at the synapse are regulated. In addition to being at the synapse, some NMDA receptors are extrasynaptic where they may have functions distinct from those at the synapse. As we reported last year, these two populations are regulated differently. It has been shown previously that NMDA receptors can bind to several PDZ proteins through an interaction with the distal C-terminus of the NR2 subunit. Of particular importance is the PSD-95 family of PDZ proteins, which includes, in addition to PSD-95, PSD-93, SAP97 and SAP102. In our earlier studies, we found that SAP102, an interacting partner of the NMDA receptor, was present throughout the neuron while its companion PDZ protein, PSD-95, was much more restricted to the synapse. Since both associate with the NMDA receptor, this led to the suggestion that SAP102 may be associated with NMDA receptors that are being transported to and from the synapse. Addressing this hypothesis, we carried out a yeast two hybrid screen using the PDZ domain of SAP102 as bait. One of the interacting proteins was Sec8, a component of the exocyst. We have confirmed that Sec8 interacts with a complex of SAP102 and the NMDA receptor. This interaction begins in the endoplasmic reticulum (ER), and disruption of the Sec8 interaction using a dominant negative construct blocks the delivery of the NMDA receptor to the plasma membrane in both heterologous cells and neurons. These results point to a critical role of the exocyst in the delivery of the NMDA receptors by way of an interaction with the PDZ protein. We are continuing this work to understand more completely the role of the exocyst in the delivery of NMDA receptors to the synapse as well as its role in the delivery of other receptors. Our preliminary results show that AMPA receptors are also changed by the expression of wild type Sec8 and dominant negative Sec8. We are also interested in determining whether or not all type 1 PDZ proteins associate with Sec8. One limitation in the analysis of receptor trafficking in neurons using tagged receptor constructs is that analyses are limited to steady state measurements. To address this limitation, we have used chimeras of GFP-tagged temperature-sensitive vesicular stomatitis viral glycoprotein mutant (VSVG) and the distal segment of NR2A or NR2B subunits. This construct is retained in the ER and then released by a change in temperature. Using this approach we can determine where various proteins associate and dissociate with the NR2 C-terminus. We find the SAP102 associates with the chimera immediately after release from the ER, which fits very well with our data on the exocyst and SAP102 association, and remains associated until the chimera reaches the synapse. PSD-95, however, has a somewhat different pattern in that it associates at the level of the trans-Golgi network. While association of the chimera with the PDZ proteins requires an intact PDZ interacting domain, this domain is not required for delivery to the vicinity of the synapse, suggesting that additional signals are involved in delivery to the synapse. Both NR2A and NR2B similarly associate with SAP102 and PSD-95. Since our research shows that associated PDZ proteins play major roles in the trafficking of NMDA receptors, we are interested in proteins that associate with the PDZ proteins and that may influence their trafficking. Using two hybrid screening, we have identified a protein that interacts with the SH3/GK domain of SAP102. This protein was previously identified and named LGN or Pins. LGN/Pins has been shown to play a role in asymmetric cell division in drosophila. Our preliminary results show that LGN/Pins influences the distribution of SAP102 both in heterologous cells and in neurons, and its increased expression appears to influence spine number and size. This may be an important protein that regulates delivery to the synapse. This past year we have completed a study on the effect of deletion of the GluR2 subunit on the assembly and synaptic delivery of the remaining subunits in the hippocampus. As described in detail in a previous report, removal of GluR2 results in the aberrant formation of GluR1/GluR3 complexes suggesting the GluR2 plays a critical role in determining the types of complexes formed in neurons. Although the expression levels remain the same, the amount of GluR1 or GluR3 at the synapse is reduced indicating that GluR1/GluR3 complexes or homomeric receptors are less efficiently delivered to, or maintained at, synapses. In analyzing synapses throughout brain, we found that the effect of GluR2 removal on remaining subunits produced two general patterns, a decrease or an increase in synaptic association. Synapses with large amounts of GluR2, and, therefore, calcium impermeable receptors, in the normal animal responded with a general reduction in the remaining subunits at the synapse. Those with normally small amounts of GluR2, and, therefore, contain more calcium permeable AMPA receptors, saw an increase in the remaining subunits. These observations raise the interesting possibility that calcium flux through AMPA receptors may play a role in the number of receptors present at the synapse.